This is a continuation of RP6 of the prior MRCE program, now presented as RP5 of a PPG (RP4-RP8) to analyze the mechanisms responsible for the broad spectrum activities of interferons (IFNs) against priority pathogens. While much is known about IFN signaling, less is known about the IFN-induced effector mechanisms that control infection. We speculate that direct manipulation of specific IFN effector mechanisms may provide broad protection without the toxicity associated with IFN administration. Our goal was two-pronged: (i) to discover novel IFN-induced antiviral molecules, and (ii) to define their importance, biochemistry, and mechanisms. We identified ISG15 as an IFN-induced anti-viral molecule, proved it's broad antiviral role, identified host proteins and ISG15 residues essential for its antiviral function, and identified a novel immune evasion strategy used by several viruses including Crimean Congo Hemorrhagic Fever Virus to counter ISG15-and ubiquitin-dependent innate immunity. While analyzing ISG15, we continued the discovery process, identifying the autophagy gene ATG5 as essential for IFNy-induced control of infection with Listeria monocytogenes (LM), Toxoplasma gondii (TG), and murine norovirus (MNV). We propose to continue this two two-prong approach to mechanism and discovery through the following Aims: Aim 1: IFN-induced effector molecule mechanisms: Focus on autophagy. 1a) Assess the generality of the importance of Atg5 and Atg7 in control of infection with priority pathogens. 1b) Determine whether autophagy proteins Atg5 and Atg7 are required for IFNa-mediated inhibition of viral growth. 1c) Define the mechanisms responsible for the importance of Atg5/7/autophagy in IFN-induced control of pathogen replication. 1d) Identify signaling mechanisms by which IFNy regulates autophagy. Aim 2: IFN-induced effector molecule identification. 2a) Define the transcriptional signature of IFNa and IFNy in primary macrophages. 2b) Screen candidate IFN-induced effector molecules for antiviral activities in vivo and in vitro. 2c) Generate and analyze mice lacking candidate antiviral molecules.